Previous work has revealed a control operative in human cells and cells of other mammalian species that represses the rate of initiation under conditions which inhibit the charging of tRNA with amino acids. The goal of the project is to elucidate the mechanism of this initiation inhibition, using both in vivo and in vitro studies of HeLa cell translation components. Crude HeLa cell initiation factors will be fractionated to identify and at least partially purify the factors required for these steps. Assays for all four partial reactions of initiation will be investigated for possible inhibition by uncharged tRNA, alone and in combination with cell fractions that might contain coinhibitors. Factors and ribosomal subunits will also be isolated from cells treated with inhibitors of tRNA charging, to determine if their capacity to function in any in vitro initiation assay has been reduced. Attempts will be made to characterize any modification of factors and subunits which are detected. The possibility that inhibition of tRNA charging might indirectly repress initiation by leading to accumulation of a compound other than uncharged tRNA which inhibits initiation will be investigated also. When the inhibition of any of the in vitro assays is observed, we will endeavor to identify the precise molecular basis for the inhibition as regards effects on the function of initiation factors, ribosomal subunits, and other molecules required for the relevant step. We will determine whether the apparent lack of the initiation control being investigated in mouse reticulocytes reflects a specific lack of susceptibility of initiation on globin mRNA to this control or, instead, a loss of this initiation control in erythroid differentiation. Translation of globin mRNA in Friend leukemia cells induced to form hemoglobin will be studied for this purpose.